Nucleotide sugars produced via enzymatically catalyzed reactions (e.g., using glycosyltransferases) and/or other synthetic methods are often obtained in the form of complex mixtures that include not only the desired compound but also contaminants such as unreacted sugars, salts, pyruvate, phosphate, nucleosides, nucleotides, proteins and the like. The presence of these contaminants is undesirable for many downstream applications. Side products are typically removed using one or more chromatographic purification steps. A common chromatographic method is reverse-phase chromatography. However, reverse-phase chromatography is often not feasible for large-scale applications due to expensive separation media and limited supply of pre-packed columns. In addition, for reverse-phase chromatography, organic solvents are typically required for optimal separation and resolution. A need exists for cost- and time-efficient processes and purification methods useful for the production of nucleotide sugars and their isolation from complex reaction mixtures. The present invention addresses this and other needs.